Role of ubiquitin conformations in the specificity of protein degradation: Iodinated derivatives with altered conformations and activities

Cox, M J; Haas, Arthur; Wilkinson, Keith D.

doi:10.1016/0003-9861(86)90742-3

Cited by 24 publications

(9 citation statements)

References 25 publications

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“…Ubiquitin has been shown to undergo an alcohol-induced transition to a partially folded state (A-state). For the A-state, NMR experiments performed in a 40:60 water/methanol solution suggested that it retains a majority of its native secondary structural elements in the N-terminal half, whereas the structure of the C-terminal half unfolds to a highly helical more elongated state. ,− For the 5 + ion sprayed from a denaturing solution, our ion/ion reaction results show that K29 and R54 are labeled (Table ), the same results as determined for the 5 + ions from aqueous conditions, consistent with CCS distribution being very similar between the 5 + sprayed from denaturing conditions and the 5 + and 6 + sprayed from native conditions. The ion/ion covalent labeling also illustrates that the peak around 1400 Å 2 in the aqueous 6 + and denaturing 5 + likely reflects compact structures, since the labeled sites are identical for native 5 + /6 + and denatured 5 + .…”

Section: Resultssupporting

confidence: 75%

Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions

Carvalho

Kit

Webb

2020

J. Am. Soc. Mass Spectrom.

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Gas-phase ion/ion chemistry was coupled to ion mobility/mass spectrometry analysis to correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross section (CCS) distributions were measured to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Aqueous solutions stabilizing the native state of ubiquitin yielded folded ubiquitin structures with CCS values consistent with previously published literature. Denaturing solutions favored several families of unfolded conformations for most of the charge states evaluated. Gas-phase covalent labeling via ion/ion reactions was followed by collision induced dissociation of the intact, labeled protein to determine which residues were labeled. Ubiquitin 5 + and 6 + electrosprayed from aqueous conditions were covalently modified preferentially at the lysine 29 and arginine 54 positions, indicating that elements of three-dimensional structure were maintained in the gas phase. On the other hand, most ubiquitin ions produced in denaturing conditions were labeled at various other lysine residues, likely due to the availability of additional sites following methanol and low pHinduced unfolding. These data support the conservation of ubiquitin structural elements in the gas phase. The research presented here provides the basis for residue-specific characterization of biomolecules in the gas phase.

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Section: Resultssupporting

confidence: 75%

Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions

Carvalho

Kit

Webb

2020

J. Am. Soc. Mass Spectrom.

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“…Recombinant E2-25K and GST-E2-25K were purified after expression in Escherichia coli (10) ), the C170S mutant form of E2-25K (10), and recombinant murine Ub-activating enzyme (E1) were gifts from C. Pickart (Johns Hopkins University, Baltimore, MD). Ub monoiodinated specifically on Tyr 59 (23) was synthesized by treating Ub (1 mg/ml) with equimolar I 2 in 50 mM Tris-HCl, pH 10.5, for 90 min at 25°C, and the monoiodo-Ub product was isolated by reverse phase HPLC (Vydac C4 column eluted at 0.75 ml/min with a linear gradient of 32-40% acetonitrile in 0.1% trifluoroacetic acid). The bovine PA700 complex was provided by G. DeMartino (University of Texas Southwestern Medical Center, Dallas, TX), and purified recombinant human UCH37, the Ub isopeptidase component of the complex, was prepared in our laboratory by W. Xu (24).…”

Section: Methodsmentioning

confidence: 99%

Cyclization of Polyubiquitin by the E2-25K Ubiquitin Conjugating Enzyme

Yao

Cohen

2000

Journal of Biological Chemistry

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For most substrates of ubiquitin (Ub)-dependent degradation, recognition by the proteasome is mediated by a covalently attached signal assembled from multiple ubiquitins linked to each other via the C terminus of one Ub and the ⑀-amine of Lys 48 of another Ub. Among Ubconjugating enzymes, E2-25K is unique in its ability to synthesize in vitro unanchored Lys 48 -linked poly-Ub chains from mono-or poly-Ub, E1, and ATP; thus, E2-25K has distinct binding sites for donor and acceptor (poly)Ub. During studies of chain assembly by E2-25K, we observed that Lys 48 -linked tri-Ub was efficiently converted to a new species that upon SDS-polyacrylamide gel electrophoresis migrated between linear di-Ub and tri-Ub. Analysis of this product by mass spectrometry and tryptic digestion showed that it was a cyclic form of tri-Ub. Cyclization of tri-Ub requires E1, E2-25K, ATP, and that the linear substrate has a free Gly 76 C terminus on the proximal end Ub and a Lys 48 side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Ub chains, including tetraand penta-Ub. Although cyclic tri-Ub resists hydrolysis by the PA700 or isopeptidase T deubiquitinating enzymes, it can be disassembled to Ub monomers by isopeptidase(s) in a red blood cell extract. Thus, if cyclic poly-Ub forms in vivo, it will not accumulate as a deadend product.

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“…John et al, 1986;Rechsteiner, 1987). The role of the carboxyl terminus of ubiquitin in its activation and subsequent conjugation of proteins has been characterized (Wilkinson & Audhya, 1981;Sobhanadity et al, 1988) and the importance of several other residues investigated by chemical modification and site-specific mutagenesis (Cox & Wilkinson, 1986;Ecker et al, 1987; Chau et al, 1989). Apart from Lys-48 and residues at or near the carboxyl terminus, however, no single residue strictly essential to ubiquitin activity has been localized.…”

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confidence: 99%

Structural and functional changes associated with modification of the ubiquitin methionine

Bamezai

Banez²,

Breslow³

1990

Biochemistry

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The effects of oxidation and cleavage of Met-1 of ubiquitin on conformation and biological activity were individually investigated. Proton NMR studies demonstrated that oxidation to the sulfone led to restricted structural perturbations at neutral pH, particularly in the vicinity of Ile-61. Below pH 3, in the presence of acetic acid, oxidation to the sulfone facilitated a conformational expansion demonstrable by retardation on gel electrophoresis and CD changes below 210 nm. The predominant phase of the low-pH transition did not involve significant changes in alpha-helix content, indicating the capacity of ubiquitin for limited structural transitions. Cleavage of Met-1 by CNBr, on the other hand, was associated with a global unfolding transition below pH 4 that involved a major loss of alpha-helix. Differences in the behavior of the native and des-Met proteins at low pH indicate that Met-1 contributes a minimum of 3.4 kcal/mol to the stability of the native conformation. Two Met-1 sulfoxide isomers, of markedly different conformational stability, were formed by treatment of ubiquitin with H2O2. One isomer was similar in stability to the sulfone, while the other was intermediate in stability between the sulfone and des-Met proteins, the differences potentially interpretable in terms of the geometry of the Met-1-Lys-63 hydrogen bond. The overall activities of the oxidized and des-Met derivatives in ATP-dependent proteolysis differed subtly from that of native ubiquitin. The unresolved sulfoxides exhibited an approximately 50% increase in activity, while the sulfone and des-Met proteins exhibited a 50% decrease in activity at low concentrations and normal activity at higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

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Role of ubiquitin conformations in the specificity of protein degradation: Iodinated derivatives with altered conformations and activities

Cited by 24 publications

References 25 publications

Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions

Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions

Cyclization of Polyubiquitin by the E2-25K Ubiquitin Conjugating Enzyme

Structural and functional changes associated with modification of the ubiquitin methionine

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